The single greatest challenge in the treatment of cancer has always been to uncover therapeutic agents that eliminate only tumor cells while sparing normal cells. The concept of synthetic lethality has much to offer here. Given the complexity of cellular networks, the best approach to uncovering novel synthetic lethal partners is from genetic screens. We recently identified the DEXH helicase, DHX9, as a synthetic lethal target from an RNA Interference screen. DHX9 is a multifunctional protein with roles in transcriptional regulation, mRNA translation, and miRNA processing. Our previous work with the RNA helicase, eIF4A, has demonstrated that selective pharmacological targeting of this class of proteins is achievable. The goal of this project is to develop HTS assays that will allow discovery of small molecule inhibitors of DHX9, which in turn, can be used in cell and animal models to examine the consequences of inhibition of this protein on cancer cell survival. We will develop a fluorescence-based, quantitative high-throughput assay to monitor the helicase activity of DHX9. In addition, an orthogonal counterscreen and secondary assay will be developed to allow triage of initial primary hits. Biochemical assays that probe the RNA helicase, RNA binding, and NTPase activities of DHX9 will be used to classify compounds based on mode of action, to provide an appropriate benchmark for specificity, and to obtain preliminary structure-activity relationships (SAR) insight. A set of cell-based assays will be developed to evaluate the effects of small molecule inhibitors on DHX9- dependent translation and on synthetic lethal relationships for triggering cancer cell death.